Detection of Plasmodium simium gametocytes in non-human primates from the Brazilian Atlantic Forest

Abstract

Background Plasmodium species of non-human primates (NHP) are of great interest because they can naturally infect humans. Plasmodium simium, a parasite restricted to the Brazilian Atlantic Forest, was recently shown to cause a zoonotic outbreak in the state of Rio de Janeiro. The potential of NHP to act as reservoirs of Plasmodium infection presents a challenge for malaria elimination, as NHP will contribute to the persistence of the parasite. The aim of the current study was to identify and quantify gametocytes in NHP naturally-infected by P. simium. Methods Whole blood samples from 35 NHP were used in quantitative reverse transcription PCR (RT-qPCR) assays targeting 18S rRNA, Pss25 and Pss48/45 malaria parasite transcripts. Absolute quantification was performed in positive samples for 18S rRNA and Pss25 targets. Linear regression was used to compare the quantification cycle (Cq) and the Spearman's rank correlation coefficient was used to assess the correlation between the copy numbers of 18S rRNA and Pss25 transcripts. The number of gametocytes/µL was calculated by applying a conversion factor of 4.17 Pss25 transcript copies per gametocyte. Results Overall, 87.5% of the 26 samples, previously diagnosed as P. simium, were positive for 18S rRNA transcript amplification, of which 13 samples (62%) were positive for Pss25 transcript amplification and 7 samples (54%) were also positive for Pss48/45 transcript. A strong positive correlation was identified between the Cq of the 18S rRNA and Pss25 and between the Pss25 and Pss48/45 transcripts. The 18S rRNA and Pss25 transcripts had an average of 1665.88 and 3.07 copies/µL, respectively. A positive correlation was observed between the copy number of Pss25 and 18S rRNA transcripts. Almost all gametocyte carriers exhibited low numbers of gametocytes (< 1/µL), with only one howler monkey having 5.8 gametocytes/µL. Conclusions For the first time, a molecular detection of P. simium gametocytes in the blood of naturally-infected brown howler monkeys (Alouatta guariba clamitans) was reported here, providing evidence that they are likely to be infectious and transmit P. simium infection, and, therefore, may act as a reservoir of malaria infection for humans in the Brazilian Atlantic Forest.

Zoonotic Malaria Risk in Serra Do Mar, Atlantic Forest, Brazil

“…In 2012, we found a surprisingly high infection rate (5.2%) of zoonotic malarias among Kerteszia mosquitoes [1]. As we knew in advance that the reemergence of the yellow fever virus in Brazil from 2016 to 2019 decimated most of the Alouatta populations throughout the Atlantic Forest [25,26], the study hypothesis was that the 2021-2022 zoonotic malaria infection rate in Kerteszia should be zero (0%) as supported by the absence of Alouatta monkeys [25,26] to locally infect mosquitoes [23]. Alternatively, if this zoonotic malaria risk was in any estimate greater than zero, other simian reservoir species might be occurring, or human hosts may sustain human-to-human zoonotic malaria transmission in the studied area.…”

“…Atlantic Forest howler monkeys (Alouatta guariba clamitans) are the main reservoir for simian malaria [7,23,37]. However, their populations steeply declined after yellow fever virus outbreaks from 2016 to 2017 [25,38].…”

“…In 2015-2016, it was suggested that every human vivax malaria case locally acquired in the Atlantic Forest may be due to a P. simium zoonotic infection [10]. More recently, it was shown that New World Alouatta simians (platyrrhine howler monkeys) can contain sufficient P. simium gametocyte density in the blood to infect mosquitoes [23]. In addition, reports showed that 18S and cytb gene sequences of P. falciparum in Kerteszia mosquitoes [1,2] and seroprevalence against P. falciparum circumsporozoite proteins in howler and other New World monkeys [24] posed a further zoonotic malaria risk due to Plasmodium spp.…”